West Nile virus (WNV) is a Flavivirus attracting worldwide attention because it has spread rapidly across the Americas since its first appearance in New York City in 1999. Several PCR-based diagnostic methods have been developed for the detection of WNV. The focus of these assays has been WNV lineage 1 which can be found worldwide, while lineage 2 viruses were thought to be endemic only in some regions of Africa. However, both lineages may be imported from Africa to Europe by migrating birds. In order to determine the incidence of WNV in Germany, a real-time-based PCR assay was developed, targeting a conserved region of WNV lineages 1 and 2. This assay is a suitable tool for the diagnosis of WNV and for surveillance studies.